The arylomycins are a series of biphenyl-linked macrocyclic lipopeptide natural products that inhibit bacterial signal peptidase I (SPase) in vitro, but show low potency and spectrum as antibiotics. SPase is an essential enzyme required for viability and virulence in all bacteria. However, after some initial interest, these natural products have been abandoned by pharmaceutical industry due to insufficient potency. The low potency of these natural product antibiotics is presumably due to their use in bacterial warfare over eons of time - most bacteria having already evolved resistance. We have synthesized one member of this class of natural products, arylomycin A2, and during its biological analysis we discovered that the mechanism of resistance in the important human pathogens Escherichia coli and Staphylococcus epidermidis is based on the introduction of a proline residue into a conserved region of the SPase substrate binding site. Sequence analysis of SPases in other bacteria reveals a remarkable correlation between arylomycin resistance and the presence of the critical 'resistance-conferring'proline residue. We have shown that bacteria that do not possess the proline, including important Gram-positive and Gram-negative human pathogens are sensitive, and that bacteria that do, are rendered sensitive by its removal. This data suggests that if the arylomycins could be re-engineered to bind SPase regardless of the 'resistance-conferring'proline, they would again be potent, broad spectrum antibiotics. In this R21 exploratory research grant, we propose to further characterize the mechanism of arylomycin resistance and use this insight to determine how the arylomycins might be re-engineered to again be potent antibiotics. More specifically, the role of the proline in the substrate binding site of SPase from P. aeruginosa will be characterized to demonstrate that this important pathogen would also be within the spectrum of a re-engineered arylomycin. In vitro kinetic data and X-ray crystallography will then be used to elucidate the mechanism of arylomycin resistance at the molecular level. Finally, after characterization of the arylomycins'mechanism of action, all of this data, along with chemical synthesis, will be used to determine which part of the arylomycin scaffold is best suited for medicinal chemistry efforts. Our project will provide a detailed understanding of the molecular evolution of resistance, and importantly, should elucidate how the arylomycins might re-engineer for potency. We hope that this, along with the fact that these compounds act via a novel mechanism of action - the inhibition of protein transport - will reinvigorate the pharmaceutical industries interest in these remarkable natural products. PUBLIC HEALTH RELEVANCE: The development of the arylomycin class of natural product antibiotics, which kill bacteria via the novel mechanism of inhibiting type I bacterial signal peptidases and protein transport, has been abandoned because these compounds are not sufficiently potent against many bacteria. It appears that over eons of being exposed to the arylomycins, bacteria have already evolved resistance. We have discovered the mechanism of this resistance, and after demonstrating that the mechanism is common among different bacterial species, we propose experiments that will elucidate how to re-engineer the arylomycins to again be potent antibiotics.